Cloning of a Putative G‐Protein‐Coupled Receptor from Arabidopsis thaliana

Abstract

We have cloned and characterized a cDNA from Arabidopsis thaliana that most likely encodes a novel member of the vast superfamily of G-protein-coupled receptor proteins (GPCRs). By taking advantage of amino acid sequence similarities between plant expressed sequence tags (ESTs) and established G-proteincoupled receptor sequences, a probe was obtained which was used for the screening of an Arabidopsis cDNA library. The cDNA which was found is very infrequently represented in the cDNA library, suggesting a low and/or spatially restricted expression. A region of the translated sequence of the cDNA shows the highest similarity to CAMP receptors from the slime mold Dictyostelium discoideurn. The same region is also similar to that in members of the animal calcitonin family of receptors. Another region of the putative receptor, however, is similar to sequences of serotonin receptors and other receptors of the socalled rhodopsin family of GPCRs. The rhodopsin family has numerous members in higher vertebrate species. Alignments and phylogenetic analyses of the regions of similarity yielded results in accordance with other evolutionary considerations. Our cDNA thus occurred on a distinct major branch in relation to the rest of the rhodopsin family. In relation to the calcitonin family, our cDNA and CAMP receptors occurred together on a distinct major branch but appear to have diverged from each other shortly after their divergence from the rest of the calcitonin family. Other features further argue for a tentative identification of it as a GPCR. It displays seven discrete and strongly predicted transmembrane domains when analyzed in hydropathy plots. The preferred orientation is with the amino terminus towards the outside. It has one Cys residue in extracellular loop 1 and another in extracellular loop 2. Cys residues in these loops are known to form disulfide bridges in many other GPCRs. Finally, it has several fully conserved amino acids that belong to the most conserved in previously known GPCRs, that occur in the above regions of similarity.

The genome sequence of the filamentous fungus Neurospora crassa

“…5), three of which belong to a new class not previously identified in any fungus. These three genes encode proteins similar to cyclic AMP GPCRs from the protists Dictyostelium discoideum 46 and Polysphodylium pallidum, and also to predicted proteins from Arabidopsis thaliana 47 and Caenorhabditis elegans. The D. discoideum proteins sense cAMP levels during chemotaxis and multicellular development 48 .…”

GCR1 Can Act Independently of Heterotrimeric G-Protein in Response to Brassinosteroids and Gibberellins in Arabidopsis Seed Germination

“…At the time GCR1 was cloned (Josefsson and Rask, 1997;Plakidou-Dymock et al, 1998), genome sequence information was limited and computational approaches for structure prediction were less sophisticated than at present. We revisited the topic of GCR1 homology to the GPCR model using current bioinformatic tools.…”

“…To date, the most promising candidate for a plant GPCR remains GCR1, independently cloned by two groups (Josefsson and Rask, 1997;PlakidouDymock et al, 1998). GCR1 encodes a protein with predicted seven membrane-spanning domains and has some sequence similarity to Dictyostelium cAMP receptors.…”

Expression Profiling and Bioinformatic Analyses of a Novel Stress-Regulated Multispanning Transmembrane Protein Family from Cereals and Arabidopsis,

“…The second GCR1, was isolated by its sequence homology with the Dictyostelium spp. cAMP GPCR (Josefsson and Rask, 1997;Plakidou-Dymock et al, 1998). These cAMP receptors are not clustered with the Rhodopsin-like family, but GCR1 possesses a motif similar to the D-R-Y triplet (H-R-T).…”